Aldehyde dehydrogenase (ALDH) is an evolutionarily conserved enzyme with pyridine nucleotide dependent oxidoreductase activity that performs a variety of critical cellular processes (Marchitti et al., 2008). These processes include production of retinoic acid essential for mammalian development (Niederreither et al., 1999), metabolism of fats and amino acids, and detoxification of endogenous and exogenous sources of hazardous aldehyde byproducts (Vasiliou et al., 2004). Twenty human ALDH genes have been identified, but many of their functions are still unknown (Black et al., 2009).
Stem cells are important new reagents in biomedical research. They can provide experimental models for target discovery (Tiscornia et al., 2011) or toxicity testing (Sison-Young et al., 2012) and can be used as tools for screening drugs and in developing therapies for a host of disorders (Ebert et al., 2012; Inoue and Yamanaka, 2011). Stem cells also can provide a direct source of materials for regenerative medicine (Cherry and Daley, 2013). The ability to prepare pure, undamaged and functionally active stem cells is the critical first step for all such applications.
Isolation of stem cells is generally accomplished by selection with monoclonal antibodies that recognize stem cell-specific cell surface markers or by utilizing functional markers of stem cell activity, such as elevated expression of multi-drug efflux pumps (Bunting, 2002) and ALDH (Jones et al., 1995). Combinations of multiple markers, which often require multiple steps of selection, are used to isolate rare stem cells from heterogeneous cell populations. A brief, single-step isolation method would be preferred to minimize loss of and damage to rare stem cells.
For the past two decades, ALDH has been studied as a potential universal marker for normal and cancer stem cells as certain isoenzymes of the ALDH superfamily have been identified as key elements of these cells (Ma and Allan, 2011). For example, Aldh1a1 and Aldh3a1 have been implicated in the protection of stem cells from cytotoxic drugs. ALDHpos stem cells have been used as resources for regenerative medicine in preclinical models (Balber, 2011) and in an ongoing clinical trial for ischemic cardiomyopathy (clinicaltrial.gov, NCT00314366). ALDH1 has been identified as a marker used to isolate cancer stem cells of various human malignancies including bladder, breast, cervical, colon, head and neck, liver, lung, pancreas, prostate, and ovary (Ma and Allan, 2011). Recently Gerber et al. showed that the presence of leukemic stem cells with intermediate ALDH activity (ALDHint) could be used as a predictor for relapse after therapy, whereas normal hematopoietic stem cells (HSCs) retain high ALDH activity (Gerber et al., 2012). Since these normal and cancer stem cells are very rare, methods to identify and isolate viable, functionally active ALDHpos cells are needed to characterize or utilize them.
The ALDEFLUOR™ reagent (Aldagen Inc., Durham, N.C.) has enabled the primary commercial assay used today for isolation of viable ALDHpos cells, which was patterned after the original dansyl aminoacetaldehyde (DAAA) based assay developed by Jones et al. (1995). Although very sensitive and specific for staining viable ALDHPos cells, because it emits in the green region of the electromagnetic spectrum (512 nm), the ALDEFLUOR™ reagent cannot be simultaneously utilized in cells or mice expressing green fluorescent proteins (Shima et al., 2012; Duncan et al., 2005). This characteristic has limited the use of many valuable cell and animal models with green fluorescent signals to study ALDH.